Falseness in the Mirna-Field As an Indicator of Strategic Bias in the Research System Via Peer-Review and Publishing Eligibility

Journal of Investigative Genomics

Mini Review Open Access Falseness in the miRNA-field as an indicator of strategic bias in the research system via peer-review and publishing eligibility

Abstract Volume 4 Issue 3 - 2017 Bias is the main barrier to a current scientific understanding. It is the tendency which prevents unprejudiced consideration of a question, and the deviation of the expected value of Roman Anton University of Truth and Common Sense, Germany the reality, and the systematic error introduced into any testing by selecting or encouraging one outcome over others. It is not only the systematic error introduced to samples–it is Correspondence: Roman Anton, University of Truth and also the systematic error introduced in any system including systems of thought and entire Common Sense, International and Darmstadt, Mangoldweg 27, research fields. The case of the miRNA (miR) research field helps to illustrate and resolve 64287 Darmstadt, Germany, Tel 491729143291, these inherent strategic biases in science that only benefit the biasing peers at the expense of Email [email protected] everyone else. Genomics especially requires an unbiased assessment of the genome and the many gene functions in a collective, free but unbiased systems biology approach with equal Received: November 05, 2017 | Published: December 19, and fair opportunity and enough jobs for PhDs and PostDocs as a relevant prerequisite. 2017 Among the many genes are also genomic microRNA loci that are processed into small non-coding but conserved double-stranded miRs. Until today, these miRs are thought to fine-tune mRNA and protein levels by degrading mRNAs or by blocking their translation and both. A closer look at this interesting class of small noncoding RNAs, however, reveals that there are not only canonical miRs and miR-seeds but also non-canonical forms, i.e. isomiRs and isoSeeds. Although canonical miRs and miR-seeds are easier to identify, the majority of miRs could be non-canonical depending on the cutoff of the definition. Senior researchers and peers have introduced strategic biases to omit the underlying complexity to make an impact, career, patents, and business, and have systematically hindered PostDocs and PhDs and their publications foryears.

Keywords: bias, strategic bias, mirna, isomer, isoseed, loci, mirbase, seed, sequence, research system, falseness, dysfunction, network, censorship, scientific, oppression, eligibility, publishing, peer-review

Abbreviations: Deep sequencing, ngs with a high number of whose publications have also increased partially exponentially, layer replicate reads; GCR8, digeorge syndrome critical region 8 gene; by layer, year after year, we find the role of small non-coding double- isomiR, the variations of mirna sequences around the published stranded RNAs. These microRNAs, miRNAs or miRs, of roughly 18- mirna sequence at mirbase, which is misleadingly referred to as gold 24bp in length, have attracted much attention of scientists and the life standard database–and due to this a major source of bias the author science industry. Today, miRNAs are still thought to regulate gene of this review claims; isomiRs, all similar miRs variations processed function at the transcript level by destabilizing mRNAs or by affecting from the common genomic loci-there can be more than one seed per the efficiency of their translation, or by an intricate interplay of both.1,2 miR; IsoSeeds, all functional miR-seeds along the miR-or isomiR- miRNAs are also defined as short double-stranded unique noncoding strand that are matching with RNA or potentially DNA in canonical and regulatory RNA sequences. Moreover, a single miR-BASE entry or non-canonical ways, via Watson-Crick or non-Watson-Crick base- widely serves as the major definition, one sequence with one miR- pairing; KO studies, knock-out studies; LOB, level of bias; miRNA, identity number that is believed to be “the real miRNA” and to exert micrornas; miRISC, miRNA-induced silencing complex; miRs, these functions by imperfect base-pairing of somewhere once-defined microRNAs; NGS, next-generation sequencing; nt, nucleotide; OTV, “seed sequences”.3 This review, instead suggests a role of isomiRs overall truth value; qPCR, quantitative polymerase chain reaction; and isoSeeds and different miR-functions and widely different miR- RPF, ribosome-protected mRNA fragment; RNA, ribonucleic acid; targets. It also claims that academic genomic research and industry RVSA, the real value of scientific achievement; TNV, true novelty innovations were somewhat biased to focus mainly on single miR- value sequences and their targets, and mechanisms in the last decade. The overseen diversity of canonical and non-canonical isomiRs Introduction and isoSeeds, different miR-functions, as well as the imperfect base- The complexity and diversity of genomics on the nucleotide pair matching and binding of miRs, has led to a guessing-bias, a don’t- (nt) level further increase exponentially on the transcript and on the question-the-dogma-bias, a make-things-significant-bias, and a have- respective modification and functional level of genes, proteins, and to-claim-as-first and first-publication-bias, among many others.4 Such RNAs, and their downstream catalytic and metabolic effects. Among publishing biases are furthered by a dominant semi-scientific peer- the many different forms of genomic and epigenetic gene regulation pressure based peer-review publication system and its procedures

Submit Manuscript | http://medcraveonline.com J Investig Genomics. 2017;4(3):68‒76. 68 © 2017 Anton. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and build upon your work non-commercially. Copyright: Falseness in the miRNA-field as an indicator of strategic bias in the research system via peer-review and 69 publishing eligibility ©2017 Anton. and all mainstream-publishing demands and criteria. This silently real-world wet lab facts has to drive the extra mile without the funding forces authors to follow previously published stereotypes and pattern and time “fuel” available for it and loses the productivity needed to be to achieve the right impact points via giving the right gut feeling in successful in this world of bad sciences-and good science of PostDocs peers and only intends to give the ‘right scientific impression’ of an is everywhere stopped by publishing eligibility, peer bias, and by an otherwise semi-scientific and often semi-false and only semi-new extreme lack of enough faculty positions.5‒9 PIs handover all of this content, both visually and in reading. This is neither a critique of early pressure to do wrong or semi-right to junior scientists, especially hypothesis building nor a critique on preliminarity of science. It is a PhDs and PostDocs to drive semi-correct publishing or no paper and critique of forced semi-wrong mainstream publishing, peer and senior out forever. These publication patterns of standardized falseness and dominance and lack of normal opportunity for PhDs and PostDocs. It standardized bias are increasingly found, increasingly misleading, and is a critique of peer and system bias and behavior that does not allow to correlate with the strategic oppression of junior scientist.5‒9 This has question any pre-assumptions–and that mainly strives to only appear led to the big success of semi-correct publishing, scientific network like very professional science but is not. Strategic bias has helped robbery of intellectual property and of the ideas and projects of PhDs research explorers and exploiters to publish semi-correct results to and PostDocs. It has given much incentives for ‘bad PI behavior’ and meet a new often only fake success-driven semi-scientific publishing makes bad publishing much easier than good and correct or adequate imperative that has taken over the entire scientific landscape in all scientific reporting: fully correct scientific reports are much more ‘risk fields and nobody did stop it like in a global Milgram-experiment in exposed’ in a world of bad science and PhD and PostDoc repression publishing that follows a misleading authority bias without feeling due to their inherent honesty, clarity, realness of progress, and the any own responsibility for the drama it causes for PhDs, Postdocs, the uncredited advancement of methods and ideas. All of this is often scientific truth and the future of all sciences. not found today and high-impact point incentives of journals have worsened all even some more. Simplification is sometimes needed but Quality assurance by peer-review is generally too biased to ever if it follows a bias or stereotype it gets problematic. function and unbiased publishing eligibility does not exist. Scientist peers have been indoctrinated, at least historically by previous and High value but less appreciated scientific contributions, i.e. with strategically by recent peer networks and participate in a by default a higher but widely undetermined RVSF (real value of scientific semi-wrong semi-scientific and semi-honest publishing in an only for finding), with respect to progress, can seem often less striking, less them beneficial swarm bias behavior. Every field thus behaves and eye-catching or less bold, or identify less optimally functioning benefits like a swarm that follows the trends and semi-false biases mechanisms, more admittedly. The scientific field much aims at a new in proposition, research, outlook, and promise. By consequence, a fake professionalism that only needs to seem professional or new. It mainstream default blockage of all new and alternative hypotheses, does not need to deliver more nor does it have to be fully reproducible true and honest scientific findings and interpretation appears that due to its grey field, which, however, has caused a game-theoretical simultaneously silences and inhibits PostDocs and PhDs, especially trap for all scientists and sciences that we are all in already – not only ambitious ones who tend to strive for a new breakthrough result, in the miRNA field but also in literally every field of science probably. innovation, and scientific or a related business career. The absolute This critique would allow preliminary findings and does allow same requirement or funding to propose semi-false grants is just one key patterns and new patterns, a storylike or story-less publishing, spartan example of it that is subsequently driving more bias everywhere. or comprehensive interpretation. But it would forbid eligibility and Concomitantly, semi-correct researching and publishing became the peer-review publishing and the recent artificial research and career secret new network standard like arbitrary decision making. These scarcities strategically placed by strategy consultancies for PhDs, essentials to be productive can now also be seen in all other fields of PostDocs and especially senior PostDocs that altogether always science-not only in the miRNA-field. The strategic bias in the miR- compels a research success at the cost of the common welfare and field is just a case example to first illustrate the major dysfunctions the Good Sciences and the quality of the scientific truth, which will in the scientific systems worldwide that hamper fair career paths and be still needed to build on any of the previous results in the future options for all scientists, and the unbiased truth in science. to make progress. It would also allow junior researchers, PhDs, and PostDocs, to publish without all of these blockades and hindrances and Discussion would ensure sustainable career paths for all scientists. The miRNA- Conflicts of interests and publishing imperatives are the key drivers field exemplifies the inherent bias in science and the problematic of semi-false scientific judgment, however, they can be found more developments for scientists. frequently everywhere in science of today. This has led to mainstream In the miRNA-Field, introductions in miRNA papers have tended scientific publishing schemes, which more and more make authors to start with: miRNAs were first discovered in C. elegans in 199310 follow the peer-review patterns in a mandatory form that only seems as a regulatory class of evolutionarily conserved small non-coding and feels intuitively right but it is semi-false, often not helpful or RNAs11 that block transcript stability and translation via imperfect reproducible, key details and honesty are missing, and this is very much miRNA-mRNA base-pairing and miRISC.2 Such text rituals continue biasing the fields. Starting with the title “X mediated by Y is controlled this way, are right by the numbers, and found in most introductions by Z” or “W-induced X promotes Y by Z”, or “X is somewhat linked but often without mentioning potential shortcomings or controversies to Y in Z”, and so on, and subsequently this seemingly standardized and which of these pre-assumptions are not fully proven by now but bioinformatics-like pattern of publication would now require several seem very likely. But could miR-functions maybe differ sometimes unambiguous ‘hard facts’ to meet expectations – but most of these or to some extent? Mainstream miR-publishing had begun: a closer publications cannot unequivocally deliver such results and too much look at the evolution of the field and its publishing might be helpful want to do so and will finally give the impression as if they would. Even and revealing Casey et al.12 These and further first-founding keystone semi-false, falsely significant up to totally fake results that still could publications and the references herein have suggested canonical be the case but never were done or done this way “-to make the entire miRNA-processing and canonical miR-functions that repress mRNAs story work”, on experiments, results or methods. Whoever sticks to the which have remained an undoubted cornerstone in the field Guo et

Citation: Anton R. Falseness in the miRNA-field as an indicator of strategic bias in the research system via peer-review and publishing eligibility.J Investig Genomics. 2017;4(3):68‒76. DOI: 10.15406/jig.2017.04.00068 Copyright: Falseness in the miRNA-field as an indicator of strategic bias in the research system via peer-review and 70 publishing eligibility ©2017 Anton. al.13,2 (and references there in) that became somewhat indubitable by (anti-null-hypothesis bias), or false-failure-bias from previous peer-pressure-bias. But could they still function differently? Although research. All this early securing R&D opportunity can be devastating several important and interesting findings and claims might have for PostDocs and junior researchers and for the world of science and been reached and published, they cannot fully resolve all of the the biotech industry, especially in the long-run that we are already in- potential roles and functions and non-functions of miRNAs without for a while as it seems. discrepancy. They found interesting roles of miRNAs and apparently One could estimate such miR-discovery projects as “too early and brought our understanding a big step further. But maybe not always biased” with regard to an analogy of Moore’s law of doubling speed in a fully correct direction. How much of it is right and fully correct in technology that can exponentially shrink costs and focus efforts-not in all of its details? only on the card-, which Moore found for microprocessor transistors Due to a bias based evolution of all scientific fields of today, initially. The learning curve also plays a crucial role in this doubling going back has generally become almost impossible by the means of speed but only if it learns without false biases. One can maybe compare mainstream publishing that follows believes and dominating views the miRNA-discovery field with the first sequencing projects of the and hereby causes subsequent peer-review biases, a major over- human genome: 10years later it would have been already extremely claim-bias, and funding biases among others. In this case, mainstream more affordable to discover, in fact, and recently especially after 2008 publishing has led to the evolution of the miR-field Casey et 12al., (NIH data). To be first can be important but can also come at a high one might assume. As a result, the field has much proceeded as if price – also for the many disadvantaged postdoctoral researchers of the functions of miRNA and their seed-binding sequences were all today6‒8 that on top of this have to compete internationally. Today, now fully identified, and all roles from basic to biomedical became 10-20years later a new picture emerges in which isomiRs and unquestionable in a field that has to steadily deliver anew, which is isoSeeds could also be a key part of the solution, like potentially achieved more easily in the mainstream with add-ons to peer-believes unforeseen roles. As a direct result of this publication bias and the creating more bias in return. Some results might be simply not the general biases in the sciences of today,4 the field had to research and case one might assume, but have manifested in the bias of a semi-false publishes frequently in this way: self-fulfilling-prophecy and a first-description-publication-bias and a a. Identify miRs in disease e.g. via expression dichotomous bidirectional null-hypothesis-bias that are all in sum very difficult to revert, next to the interesting and important part of b. Find their targets, as a mainstream research strategy the primal findings of this miR-gene function ‘screening’ of the entire field. It mainly turns problematic, if alternative important hypothesis c. Gnerally do not doubt or research the core mechanism again. and projects, if PhDs and PostDocs are hindered. Such projects But there must be more flexibility in research and a new way of are not funded due to this bias, peer beliefs, and overwhelming thinking and trying out. literature in all Life Science disciplines, Biochemistry and Molecular To tackle the status quo is risky and in many contexts not allowed, Biology, Cell Biology, Oncology, Cell Biology, Genetics Heredity, like questioning authority, due to daily training in obedient behavioral Science Technology, Biotechnology and Applied Microbiology, every day for most of all people and researchers, unopposed biasing 12 Research Experimental Medicine, and so on Casey et al., seemingly effects already knows since Pavlov and Milgram. Also, the status quo generally very much based on adhering to the common believes in in research, like a real political topic, is risky to criticise still in all all fields. Thereby, strategic bias protects the dominators of the field countries. Especially in light of senior networking and the systemic from falsification and new dogmas, while all other singular and oppression of most junior researchers,6‒8 which seems to work via different claims are simultaneously not further verified, recognized default static and capriciousness-like dynamic network control of or reproduced as peers mainly follow and reinforce the mainstream all research projects and research objectives and funding. For most bias-a double-whammy-bias everywhere. junior researchers, PhDs, and PostDocs, it was not possible to aim Subsequently and consequently, miRNAs were already researched for new breakthroughs even if it is clearly the next logical step; who for many hundreds ofmillions, in € and $, and published in many gets the opportunity to work on it defines the success that is built countries Casey et al.,12 mainly in the USA, China, Germany, UK on the ideas of those who did not get the opportunity; top ideas are and Italy, in both diagnostics and therapeutics, already much before also immediately reserved and frequently ‘transferred’ to seemingly the basic canonical and non-canonical miR-functions were fully privileged researchers. Still, PhDs and PostDocs always have to characterized, resolved and entirely understood. This might have been be transparent at any times and must present all their new findings a too premature investment prioritization as one still tends to look in progress reports without the right to publish them. Some senior to some extent for the wrong things, i.e. wrong read-outs, assuming networks lead into a strategic asymmetric information bias that other basic mechanisms, and biomedical effects. Critiques are often can harm PhDs and PostDocs by hindering them, individual per 6‒8 turned around or considered immature if they reveal shortcomings individual, and does so in many cases and until today-despite all or criticise how funding is done today. Realistic views often don’t of their excellent work. Seniors in general, when biased by default, get the money needed to be mature: maturity seems to correlate with often just want or wanted their home-run with a new interesting falseness as many kids might have already guessed early in their miR and a touchdown on a promising target-no matter how-in bias- life. But why would the world demand semi-correct sciences? Early static projects by the numbers. No matter which isomiR, no matter investment in funding of biased R&D-proposals can only temporarily the shortness of seeds, no matter the costs to science and for all be beneficial and is in tendency not fully sustainable by accelerating PostDocs – just key figures and ‘something publishable to publish, new cherry-pick-based findings, results, patentable ideas and alike, which could cause a bias. Even if the peer-reviewer is not biased – or help to identify promising new things. However, these are maybe simply assuming that he or she is, or will be, or might be, already very not followed up correctly due to the too early stage costs and field many biases the general author ex ante in many cases. The bias will biases. Hence, those projects will often be stopped and a follow-up usually be followed, no matter what the intimidated junior researchers is not possible due to many things like confidentiality agreements of worldwide would say. Blockades in academia, it is difficult to escape industry research and not publishing of negative results in academia into industry, as PostDocs are recently also discriminated on the entire

Citation: Anton R. Falseness in the miRNA-field as an indicator of strategic bias in the research system via peer-review and publishing eligibility.J Investig Genomics. 2017;4(3):68‒76. DOI: 10.15406/jig.2017.04.00068 Copyright: Falseness in the miRNA-field as an indicator of strategic bias in the research system via peer-review and 71 publishing eligibility ©2017 Anton. job markets due to organized-crime-type strategy consulting firms in earlier years3 when the microRNA field and our understanding but that strategically bias HR-criteria against PostDocs to keep solution- also our misunderstanding and miR-bias were just forming. miR-over finding competitors away from firms by creating artificial scarcity for simplifications might have taken place precipitating in the thinking IQ-jobs in firms; PostDocs would also be the better consultants. of the peer network and miR-industry. Our understanding was maybe too early to firmly established with many final views and biases ever Just in case juniors are still brave enough to say something to the since the first publication-when most of the deep sequencing and NGS PI or supervisor when a red line of science is once again crossed by data was still missing, preliminary or simplified in miR-Base without the field or lab, generally speaking, they would often risk much by giving obvious good reason or explanation for most if not all entries simply mentioning it. We all have to adjust, all of us. But this leads and miR-sequences. There is a miR-Base data entry bias and we do not automatically to an adjustment-bias that can be strategically controlled have access to the site that defines the field. All decisions in sciences by stimuli from the outside of the lab-and that is what happens. All are now made behind closed doors. As a result of this bias, (I) miR- researchers of these decades, as it seems, tended to be not free of all identity and (II) seed sequences and their target specificity, and (III) the biases and are somewhat constrained to drive new projects without molecular mechanisms have been incompletely assessed. They were bias, and biasing pressures, or without further researching a potential often researched without comprising the possibility of a relevance of bias.4 This has led to a biased publication landscape of probably isomiRs or isoSeeds in the laboratory and in bioinformatics. Weren’t many hundreds or thousands of potentially unreproducibly identified they? or predicted miRNA-target genes and mechanisms in probably most model organisms, tissues, and cells. So it is obviously difficult to miR-identity bias assess how wrong science has actually gone, and how much of it is actually right, what is underrepresented and missing, and which The abundance of isomiR-sequences makes it difficult to reveal results to believe, and in which way to correctly interpret them to which of them are the right miRNAs, i.e. isomiRs, that bear a biological extract the real information again. There are of course also many function and relevance and so on and if ‘isomiR-trimming ‘could be interesting right findings and results but all leads to the question: a relevant new miRNA-mechanisms. Everybody has blindly believed Is our understanding significantly shaped by representation bias in miR-Base and related mainstream biased information donors, and a all sciences? Nobody can check all this either. To falsify or verify whole little industry network was designing miR-qPCR-systems, potentially biased results or hypotheses is often a big no-go in most NGS, and chips, for such miRNAs that were all semi-wrongly defined labs due to reasons of cost and time, at same, new hypotheses have only on the isomiR-MIMAT nt-level, no matter which firm: major it difficult. A system of all hypotheses and alternative hypothesis to life science firms were taking the miR-Base downloadable sequence verify and falsify is missing. Systems Biology is only a trendy word information, which was called “the gold standard” and wanted to make by now without a further prioritization. It has become difficult to find business this way. Nobody took much care of an isomiR-diversity that anything on Pubmed too and a systematic order is missing everywhere. is findable today all over the place e.g. in miRSeq data as the products And in the lab, even if a junior or senior researcher spends money and of the genomic miR loci. Subsequently, most researchers were not time and reveals a difference to the mainstream publication bias, it allowed to doubt anything any longer about it and some lost their will be often not worthwhile and too difficult to publish such news in jobs if they wanted to use a more precise and differentiated method to the widely selectively biased editor-and peer-review community that validate isomiR expression or function. The research field and miR- mainly favors interesting news that must be affirmatively in line with innovation industry thought they knew already the facts of the miR- recent believes and biases. To show how it really is or works doesn’t sequences and made patents and kits based on a one annotated miR- get the score. Clearly, nobody checks for all of these bias issues and Base sequence but all isomiRs were often not incorporated enough tries to normalize it. There are not many brave enough to mention nor was only one sequence to be found as “the-miR-sequence”. Until them in this network of dependency that profits from bias and from today, these sites misleadingly suggest a reliable identification of miRs 3 eliminating postdoc martyrs that would otherwise become new big based on high coverage and high replicas of deep sequencing reads. shots and leading top experts– this bias is thus also about the early But this high amount of experiments and reads, and reads permillion, elimination of scientific competition, and to control all options also can be still misleading and biased if it only suggests one unique in the field of the R&D industry. Nobody stops such strategic bias miRNA sequence with a MIMAT-ID. Already a brief look through the developments that start with the position. deep sequencing data reveals a diversity that cannot be simplified so much and so easily in only one entry. Moreover, if you do your own Back to the miR-field: subsequently, eye-washing bioinformatics NGS or deep sequencing experiments as a biological and technical approaches have emerged in the mainstream bias that has generally replicate you will often find that it only generally resembles these data tried by system and algorithm to reveal and predict miR-targets based only as a good overlap of isomiRs or similarity but also with striking on the published miRs-sequence, the published seed sequence, and differences in quality and quantity. Furthermore, there seems to be selected mRNA-targets. miR-Base serves here as both, a helpful but no good way chosen to standardize these NGS data results so far on also misleading self-styled gold-standard-like miRNA-database,14 several levels. If one compares different tissues and cell types and a miR-registry that follows its own semi-approximating annotating miR-methods one will also find isomiR-quantities that will vary even rules and normalizations that are used by most miR-scientists in the more. This also shows that the normalization must be wrong. field of today. Especially when it comes to miR-sequence definition, and which is recently widely used in its latest version 21.3 There seems But nucleotides and multiple-nucleotide differences of isomiRs to be no right way to pick one miR-sequence, in fact, most if not all can or could make a big difference, in qPCR-assays as well as in miRs is isomiRs. Which isomiRs or research would be the question biology-even one nucleotide could in theory. This cloud of nucleotide to be resolved in the first place by scientists? The standard has maybe variation in length and sequence does not only stem from genome achieved more comparability but at a too early time point when we and transcript processing, but also from editing, tailing, modifying, still do not know if there is a difference in all isomiRs. The latest trimming, shifting (5’ or 3’), substituting and additional mechanisms version of miR-Base now includes more important deep sequencing that lead to this prevalent diversity around one miR-sequence. It data, which was less and totally missing before or more incomplete spreads like the variations on a common theme around a core overlap

Citation: Anton R. Falseness in the miRNA-field as an indicator of strategic bias in the research system via peer-review and publishing eligibility.J Investig Genomics. 2017;4(3):68‒76. DOI: 10.15406/jig.2017.04.00068 Copyright: Falseness in the miRNA-field as an indicator of strategic bias in the research system via peer-review and 72 publishing eligibility ©2017 Anton. sequence that does not necessarily and not always represent the on miR-BASE might falsely imply an unbiased and standardized unique miR-BASE entry-likely most frequently not. This mass in representation of isomiR-abundance, which is always biased but to miR-diversity, i.e. isomiR diversity, is mainly found on both the 5’ which extent? MIMIAT-sequences are widely taken too early as the and 3’ strand. Also, the star-strand was initially defined as the opposite final finding by researchers and method innovators. Disturbances of strand of the active miR-repressor strand. Subsequently, it was found molecular quantification methods by isomiR abundance have not been that this star-strand could also exert a repressive activity – again checked and were not allowed to be checked rigorously. Finally, miR- indicating an incomplete and biased understanding in the miR-field identity is a matter of isomiR-quantity and quality and a pool of miR- that has manifested to early. For instance, if strands can switch-why variations is to be studied and not one miR, which would be to cost should seeds not be able to switch-which is now termed isoSeeds, i.e. and time intensive if it is done too early. seed variations per miR that could differently match a mRNA-target, to have a term for it. The number of isomiRs and their diversity will be miRNA-seed sequences and specificity bias even higher from tissue to tissue and cell type to cell type, and miR- Most and major parts of the isomiRs are constant, often 3 BASE standardization will be even more difficult and semi-falsely evolutionarily conserved, and famous miRNA-researchers have early normalized. This makes miR-Base still a great helpful tool but also a on claimed a so-called “seed sequence” of 6 to 8nt that is found 5’ of misleading one at the same time, and the last feature is the problem. the miRISC-engaged miR-strand. This seed is usually defined from Conserved elements stay the same but striking amounts of variants nt-position 2 to 8. These canonical miR-seeds were easier to identify cannot be simply omitted by simplifying into one unique MIMAT-ID due to several reasons: they have a simple evolutionarily conserved sequence. But exactly this has been done and was taken as a given by continuous seed core that could be easily found in the less conserved many in the research field and also by the industry. It has helped to 3’-UTR counterparts of mRNA transcripts. Binding of miRISC–the sell the first generation of miRNA-qPCR-kits that PhDs and PostDocs miRNA-induced silencing complex–is intended to block translation had to use without being allowed to question their meaningfulness in or to represses and reduces transcripts via transcript stability and half- light of ubiquitous isomiR abundance everywhere in all tissues and life values t1/2 supposedly by polyadenylation, decapping and 5’-to-3’ cells for most miRs with important variations and diversity in most exonucleolytic decay.2 This idea initially goes back to the previously of all cases. QPCR-data are often used to validate the expression of discovered siRNA mechanisms and reports that many miR-seeds were chip or NGS data – but most qPCR is very nt-specific and very nt- identified with striking complementarity to mRNA transcripts.15 sensitive but in a very different way than NGS and chip methods. Thus, isomiRs can interfere with validation via both specificity and This is, however, just a matter of statistics in genomics. In other non-specificity, especially if they are the most abundant miRs in words, even if there were not one real target of a specific miRNA the sample. Hence, validation of miR-expression via qPCR can be you would always find tens of thousands of potential false-positive hampered by a higher isomiR-abundance and leads to a bias already targets due to the shortness of the seed sequences: 1 nucleotide on the molecular method level, the same holds true for chip and NGS makes 4, 2 gives rise to 16, 6 gives rise to 4^6 which is 4096 a very data methods but differently. low number in comparison to the human genome of 3,2 billion or the human transcriptome of ca. 32million base-pairs, approximately Faithful, most projects followed all biases and shifted the entire 1% of the genome, which makes roughly 4^32Mb=10^10^7 5’-to- field into a zone of miR-identity, validation bias and functional 3’ directional mRNA-sequences possible. Like a restriction enzyme bias, which was all the beginning and core of many projects. My that cuts a 3+3=6 nt palindrome, the miR-seed would also statistically postdoctoral qPCR miR-validation data could also show that isomiRs bind every 4096 nucleotides on average–given randomness. Thus, the affect the validation rate but I was not allowed to publish these transcriptome has 7800 times more sequences in length than needed important methodological findings, which would have questioned the to find a 6bp-seed match. One miRNA with a 6-bp-seed would already utility of qPCR validation kits. Like with most of my key projects match with thousands of targets (7800), which is unlikely to ever I was hindered to complete and publish them as a PostDoc (hence, result in a specific effect of a miRNA that could have evolved over data not shown), starting with the confidentiality agreements that most time. Hence, where does a higher specificity arise? PhDs and PostDocs must sign these days. In summary, whenever isomiR-abundance was high the qPCR-validation results tended to be Even a long 8-nt-miR-isoSeed that binds a target mRNA with less significant e.g. when aiming at a qPCR-validation of a chip-result, 8 nts would still bind to every 65536 random nts: 488 times in the and in comparison with independent NGS data too. Still, this is the transcriptome. The question of specificity arises but was quickly set 15 tandem method of validation in use which is found in many miRNA- aside again after the papers of the Bartel laboratory came out. To publications, and isomiR-abundance is often neglected due to costs achieve hundreds of mRNA target genes it would require a seed of 8 and time. Understandably, but this can be contra-productive, as the conserved canonical core-seed nts, which is already relatively long goal remains to identify the real isomiRs that bear the biological role compared to publications of specific miRNA effects on PubMed 2017. 1 and the relevant expression–and to understand if there might be often Hence, it was claimed that seed length correlates with efficacy but not differences of isomiRs-which would make them more important. In always. Single-stranded RNA is more flexible than double-stranded summary, to understand the effects of miRNA expression one cannot DNA and this further increases the binding likelihood and would put speak of one miRNA or one of two miRNA strands-one should keep even more emphasis on this reoccurrence of an old question: How in mind that it is a cloud of isomiRs and often not one sequence. The can a miRNA-mRNA target-specificity arise and is there a hidden relevant miR might be a different isomiR and actually, all isomiRs role of miRs on genomic DNA and protein-interactions? Not every must be validated at least high-level one, which is still too costly today. potential transcription factor binding site binds a transcription factor Better validation methods for isomiRs can be innovated (methods not in the genome. But base-pairing can be more sequence-specific shown). Quantity comes into play, but as validation still partially fails than most protein-DNA or protein-RNA interactions. RNA behaves it is only a first semi-quantitative guess that seems to be grounded on different structurally than DNA. RNA and DNA are nearly identical: biases and remains isomiR-, context-, tissue-, und method-specific. RNA has a ribose and DNA a desoxyribose making it more rigid and Importantly, most graphical and statistical representations, also a uracil instead of a thymine base. As a result, dsDNA is a B-helix

Citation: Anton R. Falseness in the miRNA-field as an indicator of strategic bias in the research system via peer-review and publishing eligibility.J Investig Genomics. 2017;4(3):68‒76. DOI: 10.15406/jig.2017.04.00068 Copyright: Falseness in the miRNA-field as an indicator of strategic bias in the research system via peer-review and 73 publishing eligibility ©2017 Anton. and dsRNA is a compact A-form under physiological conditions – this and miR-target prediction are correct? Probably nobody and more could allow proteins to structurally discern between the two-and the and more recent papers have mentioned correctly that bioinformatics single-stranded versions are structurally very flexible and can bind went very wrong and misleading. Bioinformatics is helpful but its via Watson-Crick and non-Watson-Crick-base-pairing. RNA resides downsides and misleading biases bear many problems. They ideally mainly as single-stranded RNA or RNA stretches with loops, with summarize our misunderstanding too next to our biased knowledge. specific and also dynamic binding affinity and binding flexibility– This bioinformatics-based-bias is also found in salary, why should helix-parts form just thermodynamically more stable sub-forms: bioinformatics PostDocs earn more than laboratory PostDocs? imperfect miR-helices might give structural codes and cues. There is actually no scientific reason for this and must go back to an informatics-driven ‘strategy consulting network’ based discrimination During miR-biogenesis a pri-miRNA is transcribed from the of Life Science PostDocs and PhDs internationally via strategic HR genome and forms single-stranded stem-loop structures that are thought bias: strategic job requirement bias in academia and industry. For to be processed by a Drosha-and DGCR8-containing microprocessor instance, only professional and transferable PostDocs-experience and complex (1:1 and 1:2 stoichiometry?) into the shorter pre-miR stem- competency is intentionally viewed negatively by a false advisory loop semi-helix structures. They are exported into the cytoplasm strategy, all other job experiences count always very much. Who else (Exportin-5/Ran-GDP) where most of them are further processed by but a consultancy could do such a crime to science globally? the RNAase-III enzyme Dicer into classical miRNAs. Factors like TRBP2 could help Dicer to cleave the miRNA stem-loop and in the Functions of miRNAs Bias next step the Ago2/miRISC complex is recruited Chendrimada et al.16 to regulate mRNA inhibition, all these mechanisms were probably The miRNA-field has maybe rushed into its established views claimed and established a bit too fast and thus could be significantly and mechanism a bit too fast-in a time of untimely breakthrough biased. discoveries. Subsequently, nobody could claim anything else in a biasedly semi-dysfunctional peer-review community. Although this The mRNA transcript repression is believed to be mediated by has led to many interesting primary and secondary findings, it has also created a new big obstacle to publish in an unbiased way. Maybe i. mRNA transcript stability and induced degradation in a as much as it has become a big obstacle to know what is true and siRNA-like mechanism. what is not of all that has been published under the peer-hierarchy- ii. Translational repression by somehow blocking ribosomes and and network-pressures and the imperative to publish, publish, that both could be an inevitable interplay.1,2 publish–irrespective of the overall truth value (OTV), the true novelty value (TNV), the level of bias (LOB) and the real value of scientific It has been suggested that mammalian microRNAs predominantly achievement (RVSA). (>84%) act to decrease target mRNA levels via RNA silencing Guo et al.13 using ribosome profiling–but the numbers could be ‘biased’ Next to inhibition of mRNA stability and translation1,2 miRNAs and still different. Conserved miRNA sites in 3’-UTR could, in could potentially have many additional but undiscovered functions fact, lead to a higher mRNA destabilization while the open reading due to the bias: some could also frame (ORF) could also block translation. These interesting assays i. Have only minimal effects as RNA degradation products, Guo et al.,13 however, cannot confirm the exact quantitative level of overall ribosomal repression, which could vary between transcripts ii. Still play a role on DNA and in epigenetics, genomics and and is always dynamic like ribosome speed and not static like RPFs transcription, (ribosome protected RNA fragments). Many miR-target effects could be artificially measures due to the ectopic, overexpression, and other iii. Fine-tune protein-stability and function; activate functions of methods used and different mechanisms might still be in play, which different proteins was neither publishable nor researchable due to the imperative to stick iv. Drive and change protein-DNA, protein-RNA, and protein- with the predominant and biased views and peer-pressures. protein interactions, With respect to isomiR and specificity, cooperativity of the official v. Act indirectly on targets mRNAs i.e. via signaling pathways 17 miRNA and all of its isomiRs has been suggested Cloonan et al. and alike Anton et al.,20 and A closer look at the details of the miRNA-publications, however, reveals, that most bioinformatics predictions mainly fail due to bias vi. Control molecular mechanisms as other classes if they are and predominantly result in semi-false or misleading predictions inseparable from miR-definitions by sequence: like passenger-, (www.mirdb.org, www.microrna.org, www.targetscan.org, and all the sno-and guide-miRs others too). No one in this field of science seems to be able to fully vii. Be cellular sequence-binders for other mechanisms like editing, assess the truth value of these predictions accordingly and correctly but mRNA decay, and alike, everyone takes this bias as an arbitrary given. PostDocs are hindered via hierarchy and publishing criteria to reveal the truth-value about viii. Contribute toward cellular-immunity and health, anti-pathogen these predictions. Biochemical experiments and HITS-CLIP Moore et defense al.18 sees a striking overlap of these 6-8bp bioinformatics predictions ix. Mediate robustness or quality of cell fate, transcription for miRs, also according to research talks on conferences, but this networks, cell cycle, communication, homeostasis, and other seems unlikely to be the case in light of the fact that most isomiRs mechanisms and isoSeed are not well defined. That HITS-CLIP and bioinformatics predictions Chi et al.19 overlap so strikingly can have different reasons, x. They could still have unforeseen additional functions that are and could bear a wishful-thinking bias: e.g. the biochemical results are not investigated due the massive bias that only few contest. only “ago-footprints” and all models stem from lets-find-the-correct- The methods that quantify expression are technically biased and seed-matches in miR-and mRNA-datasets. Who says that all seed yield different and contradicting results, like the methods that analyze

Figure 1 Canonical and non-canonical mir-isoseeds, specificity via unconventional basepairs matching?

Conclusion avoid them. Only a scientific system that works in theory can work in practice. Systems Biology only works if we are doing it together- Despite the false pre-assumptions in the field about (a) only one without excluding or repressing scientists. unique miR-sequences instead of pools of isomiRs, of (b) only one seed instead of multiple isoSeeds, of (c) only one main function Acknowledgements instead of many functions, of (d) only one often false positive and false negative bioinformatics approach instead of promising ideas I acknowledge University of Truth International to support me and new algorithms and ways to potentially find the right targets, the in this work that was previously censored by PIs and peers and the field still has-after all (a-d)-identified interesting key mechanisms and research system by strategically blocking most postdoctoral reports many miRNAs with a potential biological relevance and biomedical and careers. impact. However, how many of these biomedical miRs that were identified in this cherry-picking approach of the science field can Conflicts of interest be verified biomedical and promising targets and candidates for Conflicts of interests are prevalent in all research of today due to therapeutic and diagnostic strategies remains to be reinvestigated due the wrong hiring practices. A sustainable research system without to the obvious biases and pressures in science of today.4,22,23 There such conflicts of interests is needed. All publications of today that do is a too high pressure on most of all junior scientists, on PhD and not mention these conflicts of interest from peer-pressures and from PostDocs who are the real scientists in the lab who do the research, what is needed for success still omit the full truth. The University of and also PIs stand under high peer bias pressure to obtain funding Truth comparable to School of Life did not financially support me in and kudos. Sustainable career paths are lacking like good jobs and any way, nor did anyone else benefit me for this review work. I did PostDocs are discriminated due to consultancies in all suitable jobs. also not receive any relieve, which in sum helped me to be harshly This increases the bias and peer pressure in science and nobody compelled to avoid any potential financial bias or dependency that ‘quality assures science’ or assures fairness for juniors and protects could inflict the unbiased nature, independence, and outcome of this it from the biases of peer-review, funding and publication biases. In work since every remuneration and financial support would anyhow order to take the next big step and to take the field to the next level, it always result in a never confessed conflict of interest and bias. Future will be important to overcome the biases in science.4,22,23 This includes job prospects were set aside only for the time of writing this review. I a correct view of the situation of all PhDs and PostDocs and to assure will have to comply with all biases to be compatible again. sustainable career paths. References With respect to the miR-field: Scientifically, it might be interesting to reveal 1. Bartel D. MicroRNAs: Target Recognition and Regulatory Functions. Cell. 2009;136(2):215‒233. a. If there are different types of seed sequences that are biologically relevant: canonical versus non-canonical seeds, 2. Jonas S, Izaurralde E. Towards a molecular understanding of microRNA- mediated gene silencing. Nature Reviews Genetics. 2015;16(7):421‒433. and multiple seeds termed isoseeds. 3. Kozomara A, Griffiths-Jones S. miRBase: annotating high confidence b. If there are different types of isomiRs that are biological microRNAs using deepsequencing data. Nucleic Acids Res. relevant: canonical and non-canonical isomiRs. 2014;42(D1):D68‒D73. c. The real or additional mechanisms involved. 4. Rothstein H, Sutton AJ, Borenstein M. Publication Bias in Meta- Analysis-Prevention, Assessment and Adjustments. Chichester, England: d. How to improve our methods to tackles all of these questions Sutton and Borenstein, John Wiley & Sons; 2005. and molecular heterogeneity, complexity and diversity of isomiRs an isoSeeds. 5. Shinbrot T. Exploitation of junior scientists must end. Nature. 1999;521(399):521. Recently, biomedical researchers and bio-informaticians have 6. Bourne HR. A fair deal for PhD students and postdocs. Elife. also correctly realized this new challenge of isomiR complexity and 2013;1(2):e01139. are eventually working on this non-canonical isomiR topic Li et al.24 The same could be done for the non-canonical seeds, and the correct 7. Holleman W, Gritz ER. Biomedical burnout. Nature. 2013;500:613‒614. algorithm to align them with mRNAs has still yet to be identified, or 8. Alberts B, Kirschner MW, Tilghman S, et al. Rescuing US Biomedical the old algorithms have to be systematically re-investigated and proven Research From Its Systemic Flaws. Proc Natl Acad Sci USA. alternatively. Canonical miRs and miR-seeds were easier to identify, 2014;111(16):5773‒5777. but the majority of miRs could be non-canonical depending on the 9. McDowell GS, Gunsalus KT, MacKellar DC, et al. Shaping the Future cut-off of the definition which is set to 2-8bp for canonical seeds due of Research: a perspective from junior scientists. F1000Res. 2015;3:291. to historical reason. Most publications highly intend to give the strong impression of science but are highly biased and pseudo-scientific. 10. Lee C, Feinbaum R, Ambros V. The C. elegans heterochronic gene lin- Everyone who criticizes them is subsequently called this way by a 4 encodes small RNAs with antisense complementarity to lin-14. Cell. dominant peer pressure bias. Many rules to avoid bias are everywhere 1993;75(5):843‒854. often not followed at all.4,22,23 Senior researchers and peers have also 11. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. intentionally introduced new strategic biases of great implications Cell. 2004;116(2):281‒297. and falseness to omit the underlying complexity and diversity and 12. Casey MC, Kerin MJ, Brown JA, et al. Evolution of a research field-a to mainly block the success of all competing researchers, to publish micro (RNA) example. Peer J. 2015;3:se829. easier, or to make their career, or to sell products, like the miRNA qPCR kits that are biased by the MIMAT ID norms, or to silence all 13. Guo H, Ingolia N, Weissman J, et al. Mammalian microRNAs junior and competitor researchers to gain an advantage and more time predominantly act to decrease target mRNA levels. Nature. 2010;466(7308):835‒840. at the cost for all and for science. Only if we research biases we can

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